This work is supported by a National Science Foundation award (1145581) to LWG.

ملاحظة: تم إجراء الإجراء بأكمله وفقا للمبادئ التوجيهية للمعاهد الوطنية للصحة، التي وافقت عليها لجنة رعاية واستخدام الحيوان من جامعة إلينوي في شيكاغو. 1. حلول والثقافة وسائل الإعلام الاستعدادات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إبقاء جميع الحلول في -20 درجة مئوية لمدة تصل إلى ستة أشهر. إبقاء وسائل الإعلام الثقافة في 4 درجة مئوية لمدة تصل إلى 3 أشهر. </li><li style=";text-align:right;direction:rtl"> إعداد 100 مل من وسائل الإعلام والثقافة عن طريق خلط 1 مل من محلول القلم بكتيريا و 1 مل من ITSX (الانسولين ترانسفيرين-مكملات السيلينيوم) إلى 100 مل مع DMEM. </li><li style=";text-align:right;direction:rtl"> إعداد الحل انزيم عن طريق خلط 250 مل من DMEM 2.5 مل من 100 مم CaCl 2، و 2.5 مل من 50 ملي EDTA. </li><li style=";text-align:right;direction:rtl"> إعداد الحل تعطيل عن طريق خلط 225 مل من DMEM، 25 مل من FBS، 625 ملغ من الزلال، و 625 ملغ مثبط التربسين. </li><li style=";text-align:right;direction:rtl"> إعداد الحل ولوك من 154 مم كلوريد الصوديوم، 5.6 ملي بوكل، 5.0 ملي HEPES، NaHCO3 3.6 ملم، 5.6 ملم الجلوكوز. ضبط درجة الحموضة إلى 7.3 مع هيدروكسيد الصوديوم. </li><li style=";text-align:right;direction:rtl">إعداد بولي د يسين (PDL) حل 50 ملغ / مل. استخدام تركيز النهائي من 1 ملغ / مل إلى coverslips معطف. </li><li style=";text-align:right;direction:rtl"> إعداد حل خارج الخلية من 140 مم كلوريد الصوديوم، 5 ملي بوكل، 2 مم CaCl 2، 1 ملم MgCl 2 و 10 ملي HEPES، هيدروكسيد الصوديوم، و 10 ملي الجلوكوز. ضبط درجة الحموضة إلى 7.3 مع هيدروكسيد الصوديوم مع الأسمولية من ~ 310 نانومول / كجم. </li><li style=";text-align:right;direction:rtl"> يعد حل ماصة من 50 ملي كلوريد الصوديوم، و 100 ملي TEACl، 5 ملي بوكل، 2 مم CaCl 2، 1 ملم MgCl 2 و 10 ملي HEPES. ضبط درجة الحموضة إلى 7.3 مع هيدروكسيد الصوديوم مع الأسمولية من ~ 290 نانومول / كجم. </li></ol> 2. الغدة الكظرية عزل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأوتوكلاف زوج من مقص تشريح الكبيرة والصغيرة واثنين من أزواج من ملقط. ارتداء القفازات أثناء العملية برمتها ووضع علبة تشريح في دلو الجليد. </li><li style=";text-align:right;direction:rtl"> استخدام الفئران التي اتخذت في يوم 0-3. الموت ببطء الحيوانات مع الضغط دبابة CO 2 تليها قطع الرأس. </li><li style=";text-align:right;direction:rtl"> الاستفادة من مجموعة أصغر من مقص لقطع أسفل الجزء الخلفي من الجرو التالية من العمود الفقري العنقية<html> كال لالذيلية المنطقة. تأكد من قطع سطحي تحت الجلد وليس بيرس في تجويف الجسم. </li><li style=";text-align:right;direction:rtl"> المقبل، قشر الجلد بعيدا عن العمود الفقري لذلك يتعرض هذا الجهاز العضلي أن يكون لها رؤية واضحة للعمود الفقري. من هنا، اجراء خفض transectional في الحبل الشوكي عنق الرحم ثم جعل اثنين تخفيضات موازية على طول جانبي العمود الفقري. </li><li style=";text-align:right;direction:rtl"> مع زوج واحد من ملقط يمسك الجسم الرئيسي للحيوان، استخدام زوج آخر من ملقط لانتزاع العمود الفقري وقشر الظهر العمود الفقري حتى تتعرض الكلى. </li><li style=";text-align:right;direction:rtl"> في هذه اللحظة، تصور الغدد الكظرية على قمة الكليتين. ثم ضع بعناية الغدد اثنين من كل حيوان في أنبوب مخروطي الشكل مملوءة بمحلول لوك. ملاحظة: في بعض الأحيان سوف الغدد التمسك ملقط. إذا كانت هذه هي الحالة، استخدم بلطف زوج آخر من ملقط للمساعدة في إزالة الغدد من ملقط والى حل للوك. يمكن أن تبقى الغدد في هذه الحالة لمدة تصل إلى 2 ساعة. </li></ol>ه &quot;&gt; 3. الهضم الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في غضون ذلك، فقاعة الحل الانزيم مع 5٪ CO 2 + 95٪ O 2 لمدة 15 دقيقة قبل الاستخدام. ضمان أن الحل معايرتها يتحول من اللون الوردي مشرق في اللون وردي / برتقالي. </li><li style=";text-align:right;direction:rtl"> إضافة غراء إلى حل انزيم فقاعات حديثا بتركيز نهائي من 20-25 وحدة / مل. احتضان كل زوج من الغدد من كل حيوان ل40-60 دقيقة عند 37 درجة مئوية في حل الانزيم مع غراء. </li><li style=";text-align:right;direction:rtl"> إضافة 75٪ من الحل تعطيل لكل أنبوب، واحتضان عند 37 درجة مئوية لمدة 10 دقيقة أخرى. هذه الحضانة يثبط نشاط نشاط انزيم غراء. </li><li style=";text-align:right;direction:rtl"> بعد حضانة 10 دقيقة مع الحل تعطيل، ونقل بلطف الغدد في أنبوب المسمى حديثا ثم يغسل الغدد 3X مع وسائل الإعلام الثقافة. </li></ol> 4. خلية التفكك والتصفيحات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد الغسيل، ووضع الغدد اثنين الى 200 ميكرولتر من وسائل الإعلام الثقافة وثم يعاير بلطف صعودا والغددإلى أسفل من خلال طرف ماصة مع ماصة 200 ميكرولتر حتى لم يعد هناك قطعة كبيرة من الأنسجة في الحل. ملاحظة: عند المعايرة، والحفاظ على الغدد في الجزء السفلي من أنبوب مع قوة الهبوط من 200 ميكرولتر ماصة وإشراك الأنسجة بلطف وببطء. تكون معينة لاستخدام بطيئة، والسكتات الدماغية مستمرة، أبدا pipetting لسريع أو مفاجئ. </li><li style=";text-align:right;direction:rtl"> إضافة إضافي سائل الإعلام والثقافة 250 ميكرولتر لتبرزي حجم الكلي للثقافة وسائل الإعلام تصل إلى 450 ميكرولتر. </li><li style=";text-align:right;direction:rtl"> لوحة 60 ميكرولتر من هذا الحل التي تحتوي على كل خلية من سبعة 12 ملم coverslips المغلفة PDL مسبقا، والتي يتم وضعها في صحن الثقافة 60 × 15 مم. </li><li style=";text-align:right;direction:rtl"> مرة واحدة مطلي، ضع الطبق في الحاضنة التي تم تعيينها إلى 37 درجة مئوية، وحافظت مع تدفق مستمر / توريد 5٪ CO 2 لمدة 30 دقيقة لضمان بيئة لبقاء الخلية. بعد الحضانة، إضافة بلطف 5 مل من قبل تحسنت وسائل الإعلام الثقافة في صحن الثقافة والعودة الطبق مرة أخرى في حاضنة لمدة 24 ساعة قبل خلية الملحقتسجيلات د. ملاحظة: عادة، الخلايا يمكن استخدامها لالتسجيلات الكهربية لمدة تصل إلى 4 أيام. </li></ol> 5. خلية تحديد وتشكيل ختم Gigaohm <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع 12 ملم ساترة إلى حل خارج الخلية. حيث يتم استخدام الغدد الكظرية كاملة لإعداد الخلية، مزيج الخلايا أليفة الكروم (عادة مشرقة جدا مع البني / البيج والتلوين على شكل دائري مثالي القريب (الشكل 1) في الثقافة مع الخلايا القشرية (أصغر وباهتة من الخلايا أليفة الكروم). </li><li style=";text-align:right;direction:rtl"> سحب ماصات التصحيح في أربع مراحل باستخدام برمجة P-97 مجتذب. تراجع غيض ماصة في الشمع المنصهر، والتي سوف تساعد على تقليل السعة من الزجاج ومن ثم اطلاق النار البولندية طرف إلى &quot;ضربة بعيدا&quot; هذا الشمع من الداخل من طرف ماصة. ملاحظة: عندما تمتلئ حل ماصة التصحيح، ماصات التصحيح وعادة ما يكون مقاومة MΩ ~ 2. </li><li style=";text-align:right;direction:rtl"> الاقتراب من ماصة التصحيح قريبة من الخلية ضمن عدة مicrons. لتحقيق تكوين الخلية المرفقة، والبحث عن أي تشوه خلية طفيف خلال نهج من ماصة التصحيح أقرب إلى الخلية. تطبيق الشفط لطيف حتى يتحقق ختم GΩ. </li></ol> 6. الخلية المرفقة التسجيلات السعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بمجرد كشف ختم GΩ، وجعل التسجيلات الخلية المرفقة من خلال استخدام مكبر للصوت EPC-7 زائد التصحيح، المشبك، وعلى مرحلتين التناظرية قفل في مكبر للصوت (انظر الجدول المرفق المعدات). تطبيق شركة Sinewave مع سعة 50 فولت (RMS) وتردد 20 كيلو هرتز من مكبر للصوت في قفل. </li><li style=";text-align:right;direction:rtl"> تعيين عامل التصفية الناتج من مكبر للصوت قفل في 1 مللي ثانية في وقت ثابت و 24 ديسيبل. تعيين مرحلة من مكبر للصوت قفل في مثل هذا أن التغيرات السعة عابرة تنتجها البقول شفط طيف تظهر فقط في السعة التصحيح (ايم) التتبع مع ​​أي الإسقاط في تصرف التصحيح (إعادة) أثر (الشكل 2). ملاحظة: يرجى الاطلاع على التفاصيل لمعايرة النظام في إعادةاملؤمتر 19. </li><li style=";text-align:right;direction:rtl"> تحديد التغييرات السعة نموذجية من خطوات &quot;الهبوط&quot; (الشكل 3). ملاحظة: سيتم شاهدوا A تسجيل الصلب بأنها &quot;الدرج&quot; نوع تشابه مع العديد من الخطوات الهبوط مما يؤدي الى استيعاب الحويصلات واحدة. تخدم عملية الترقيع بمثابة التحفيز الميكانيكي، نظرا لأن الإجراءات الحالية يمكن عادة المسجلة من معظم الخلايا. </li><li style=";text-align:right;direction:rtl"> تسمية الملفات والمجلدات الفردية بما في ذلك تاريخ: شهر / يوم / سنة وكل تسجيل الخلية المرفقة والخاصة ترقيم فردي (س س) داخل هذا الملف. </li></ol>

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تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

قياسات السعة الخلية المرفقة تتطلب عدة خطوات حاسمة من أجل الحصول على التسجيلات ذات الجودة العالية بنجاح: 1) خلايا قابلة للحياة وصحية أعدت من الغدد الكظرية. 2) PDL طلاء لل coverslips. 3) تشكيل ختم gigaohm. 4) مستوى الضوضاء النظام؛ و 5) مرحلة التصحيح. <p class="jove_content" style=";text-align:right;direction:rt...

وتوسط انتقال متشابك بواسطة إيماس من الحويصلات التي تحتوي على ناقل عصبي متشابك، ويجب أن تخضع هذه الحويصلات إعادة التدوير التقامي المحلي داخل محطة العصبية للحفاظ على الاتصالات العصبية على المدى الطويل. نظرا للدور أساسي في انتقال متشابك في الدماغ، وفهم الآليات الجزيئية التي تشكل دورة حويصلة متشابك هي الركيزة الأساسية نحو فهم أفضل في مجال الاتصالات الخلوية ككل. بين النظم نموذج خلية، وقد وفرت الخلية أليفة الكروم الكظرية بعض البصيرة الأكثر حسما في الآلية الجزيئية الكامنة وراء إعادة تدوير حويصلة متشابك. إيماس، فإن الخطوة النهائية في إطلاق الناقلات العصبيه، وقد درس كثيرا وفحصها من خلال استخدام الخلايا أليفة الكروم الكظرية 1،2. في الواقع، معظم اللاعبين الجزيئية التي تنسق تشكيل والاستهداف والالتحام والانصهار من حبيبات إفرازية تم تحديدها نظرا لتطبيق شعبةتقنيات في الخلايا أليفة الكروم ERSE 1. وعلاوة على ذلك، من خلال توفير فرصة للسماح للقرار حويصلة واحدة من الآلات البروتين المشاركة في إيماس، تبقى الخلايا أليفة الكروم نموذجا قويا لمعالجة مسائل الانصهار الحويصلة 3. واستخدمت قياسات السعة الخلية المرفقة أولا في حل واحدة الانصهار الحويصلة خلال إيماس 3. إيماس حويصلات صغيرة مثل ~ أثبتت 60 نانومتر في القطر ليتم الكشف عنها بواسطة القياسات قبول غشاء الخلية مع تقنية المشبك التصحيح في الخلية المرفقة التكوين 4-7. ويعرف قبول كمقياس لمدى سهولة الدائرة أو الجهاز سوف تسمح لتدفق الحالية. هو معكوس مقاومة. وبالتالي، توفر قياسات قبول فهم السعة الغشاء. ويتم إنجاز هذا عن طريق دمج الغشاء الحويصلي في غشاء البلازما. هذا التأسيس يكشف عن تغيرات في سطحمنطقة 8. تسبب كل حويصلة التفجير زيادة تدريجية في السعة غشاء 9،10. بالإضافة إلى ذلك، يوفر هذا القياس القبول في تصرف الغشاء والمسام الانصهار تصرف خلال هذا الحدث exocytotic 3. كما وفرت هذه التقنية أداة فريدة من نوعها في تحديد حركية حويصلة واحدة خلال إيماس، مختبرنا وقد طبقت مؤخرا هذا المفهوم للكشف عن الإلتقام من الحويصلات واحدة 11،12. مصلحتنا هي محددة بوساطة بالكلاذرين الإلتقام (CME)، والتي تم اعتبارها مكونا أساسيا في التدبير المنزلي العديد من الخلايا 13 و كمسار الرئيسي لحويصلة متشابك الإلتقام في المحطات العصبية 14،15. ومن المعروف CME إلى أن تكون مهمة من الناحية البيولوجية، ومع ذلك، لا تزال حركية ليست مفهومة بشكل جيد بسبب القيود التقنية في رصد الأحداث التقامي المفرد. نظرا لأوجه التشابه في آليات exocytic بين الخلايا أليفة الكروم والخلايا العصبية 1، فمن ررausible التي قد تطبق آليات انشطار الخلايا أليفة الكروم في الأرجح إلى حويصلة متشابك الإلتقام في الخلايا العصبية. وقد استخدمت قياسات السعة الخلية المرفقة لرصد الأحداث التقامي الفردية وتحليل حركية الانشطار، والتي معظم الطرق غير قادرة على حلها. في منطقتنا التسجيلات الخلية المرفقة، وفرضه موجة جيبية عند 20 كيلو هرتز حول احتمالات عقد، ويتم فصل الانتاج الحالي في غشاء تصرف في قناة واحدة وغشاء السعة في قناة أخرى من مرحلتين قفل في مكبر للصوت 16- 18. من تغيرات في غشاء تصرف والسعة، يمكن للمرء حساب حركية الانشطار المسام، والتي تتطابق المرجح أن الرقبة غشاء أنبوبي الذي يربط حويصلة استيعاب لغشاء البلازما قبل حويصلة قرصة حالا. جماعي، هذه التقنية تتيح لنا الفرصة لدراسة الآليات التنظيمية للحويصلة الانشطار خلال التعليم الطبي المستمر.

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			<td style="width:88px;">Outer diameter-1.5 mm</td>
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methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

وبقاء الخلية ونوعية ختم gigaohm حاسمة في تحديد نوعية التسجيلات السعة الخلية المرفقة. لذا، فمن الأهمية بمكان لشراء ثقافة خلية فعالة وكفؤة قبل التسجيلات الكهربية وخلايا قابلة للحياة نموذجية موضحة في الشكل 1. سوف الممارسة والوقت يكون مفيدا في تحقيق ختم gigaohm ذات جو...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

طرق الخلية المرفقة قياسات السعة في ماوس الكظرية أليف الكروم خلية

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

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Neuroscience

8.7K Views.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Video: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

الموجات فوق الصوتية ، إذ تسترشد Microinjection في الدماغ المقدم ماوس في الرحم في E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

متعددة الماوس تشريحي عصبي التصوير بالرنين المغناطيسي

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Electroporation في الجسم الحي من الشبكية ماوس النامية

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

التصحيح ، المشبك قياسات السعة والكالسيوم 2 + التصوير في محطات عصب واحد في شرائح الشبكية

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

أساليب الإدارة الذاتية في الوريد في نموذج الفأر

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

طرق لتعديل وتحليل NF كيلوبايت التي تعتمد على تكوين الخلايا العصبية الكبار

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

في القياسات الكهربية فيفو على الأعصاب ماوس الوركي

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

التصوير حية من الانقسام في تطوير الجنينية الماوس اللحاء

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

طريقتان للتقشير لعزل مقصورات خلايا المستقبلات الضوئية في شبكية العين للفأر لتحليل البروتين

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

الطرق الكهروفسيولوجية لقياس مستويات التصبغ الضوئي في مستقبلات ذبابة الفاكهة الضوئية

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...